Recent research has shown that nitric oxide (NO) is a vital biological molecule. NO plays a central role in such diverse processes as host defense, cardiovascular regulation, signal transduction, neurotransmission and wound healing. The enzyme nitric oxide synthase (NOS) converts L-arginine into L-citrulline and NO, and numerous cells involved in the wound healing process have shown NOS activity. The exact functions of NO in tissue repair have not been established, although a likely major role of NO is that of a cytotoxic or cytostatic agent released by macrophages and other phagocytic cells during the early inflammatory phase. NO released from wound resident cells may also be important in unique cell signalling pathways and the re-establishment of the microcirculation as newly vascularized tissue is formed.
Oxidation of NO produces unstable intermediates (such as N.sub.2 O.sub.3 and N.sub.2 O.sub.4) and subsequently the stable metabolic products nitrite (NO.sub.2) and nitrate (NO.sub.3). Previous studies have shown that urinary NO.sub.2 is negligible in wounded or infected rats and that urinary NO.sub.3 is an accurate indirect measure of NO production.
Previous work has shown that urinary NO.sub.3 levels in normal excisionally wounded rats rises sharply upon wounding and remains significantly elevated over the course of tissue repair for up to 18 days following external wound closure. However, two common impaired wound models, steroid-treated rats and experimentally induced diabetic rats, both showed suppressed NO synthesis during wound repair. This suggests that the metabolism of NO by functional biological cells may be critically important during tissue repair. Furthermore, topical application of the NOS inhibitors N.sup.G -monomethyl-L-arginine (LMMA) and N.sup.G -nitro-L-arginine (LNA) significantly reduced NO synthesis (P&lt;0.05) in wounds of normal rats, demonstrating that topical application of therapeutics can alter normal NO metabolism. If insufficient NO synthesis at the wound site is a key factor in impaired wound healing, then controlled topical delivery of NO to the local wound environment may be a new therapy for accelerating the healing of both chronic and normal wounds. Topical NO delivery may also be a crucial component of a new generation of wound dressings, since few controlled release drugs are currently available.
Recently, complexes formed by reacting nitric oxide with certain nucleophiles have been introduced as a new class of NO-releasing compounds. Keefer and coworkers have synthesized zwitterionic polyamine/NO adducts referred to as NONOates. In U.S. Pat. No. 5,250,550, Keefer et al. shows the following nitric oxide-polyamine complexes with pharmaceutically acceptable salts thereof as useful cardiovascular agents: ##STR1## Alternatively, U.S. Pat. No. 5,212,204 to Keefer et al., describes antihypertensive compositions and a method of lowering blood pressure in mammals, wherein the active component of the composition is a compound of the following formula (IV) ##STR2## wherein J is an organic or inorganic moiety and M.sup.+x is a pharmaceutically acceptable cation which does not render the compound unstable or insoluble in water.
Previous Keefer patents, e.g., U.S. Pat. No. 5,208,233 also discussed anti-hypertensive compositions and methods of lowering blood pressure in mammals, can be characterized as shown in the following formula (V) ##STR3## wherein an ionic-type association was shown, and wherein when R.sub.1 and R.sub.2 were bonded together, the following groups (VI-1 through VI-4) were preferred: ##STR4##
Additional uses for NO were shown in U.S. Pat. No. 5,185,376 wherein platelet aggregation inhibition in vivo was shown with physiologically compatible compounds containing at least one N-oxo-N-nitrosoamine moiety in a molecule thereof, wherein the physiologically compatible compound released nitric oxide in a sustained and controllable fashion in vivo. The types of compounds listed for this application were DEANO (VII) ##STR5## and the nitric oxide addition product of the polyamine spermine (VIII); ##STR6## NIPRIDE (nitroprusside), formula (IX); ##STR7## and ASA (aspirin), formula (X). ##STR8##
These NONOates release quantitative amounts of NO in aqueous media, and the rate and extent of NO generation appear to depend on pH, temperature, and the identity of the nucleophile residue
NONOate compounds can be used as NO-based vasodilators, and may have other clinical applications, such as anti-tumor therapy. However, delivery of NO to wounds via these soluble amine-based NONOates is complicated by the solubility of toxic amines that remain after NO is released.
This invention involves polymer/NO adducts that are insoluble, non-toxic, and exhibit a long, controlled release of NO in aqueous solution to create a new class of NONOates. The polymeric NONOate, NO-polyethyleneimine cellulose (PEIC-NO), releases a significant amount of NO over a long period of time in an aqueous environment. PEIC-NO was chosen for wound healing studies due to its low toxicity, ease of application, and relatively long half life (approximately 960 min). Furthermore, another cellulose derivative, carboxymethyl cellulose, is a major component of most existing hydrocolloid wound dressings, and therefore incorporation of PEIC-NO into commercially available dressing formulations should be feasible.